Chapter 3: Eucaryotic Molecular Biology, Cellular Hurdles, and How Viruses Hojack Host Cells

Genes Required for Assembly of Infectious Virus Particles

• Genes that code for:

  • capsid protein(s) - protective shell

  • virus specific enzymes (RdRP, RTase, or lysozyme)

  • viral receptor binding protein used for attachment to animal host cell

• Genome length varies greatly depending on composition and encodes 3-200 genes

  • RNA viruses: 1.7-30Kb

  • DNA viruses: 5-200 Kb

  • Giruses: 1.2 Mb

• viruses cannot reproduce except by means of resources within living cells:

  • obligate intracellular parasites

RdRP

helps RNA viruses copy their genetic material

RTase

(in retroviruses) which converts RNA → DNA

Lysozyme

helps viruses infect bacteria by breaking their cell wall

Directionality of DNA/RNA

• copied and read in 3’→5’ direction

• synthesis in 5’→ 3’ direction

Central Dogma

Think of cell as factory:

1. DNA is an instruction manual

2. RNA is copy of instructions

3. factory uses RNA to assemble proteins

The human genome

• 3 billion base pairs

• protein coding sequences (1.5%): genes

• non-protein coding sequences:

  • introns

  • promoters and enhancers

  • telomeres

  • non-coding RNA’s:

    • rRNA’s, tRNAs, microRNAs, snRNAs

RNA polymerase I

transcribes most ribosomonal rRNA

RNA polmerase II

transcribes:

• pre-mRNAs - precursor to messenger RNAs

• snRNA’s- nuclear RNA’s

RNA Polymerase III

transcribes:

• tRNA’s - transfer RNA

• 5s rRNAs

RNA Splicing

• introns: non-coding sequences

• exons: coding sequences

• splicing occurs in nucleus

• introns are removed from pre-mRNA and exons are connected into a continuous mRNA

• after splicing on 5% of originally transcribed mRNA exits the nucleus

• first discovered in HeLa cells infected with adenovirus 2

Eukaryotic Translation

• 3 steps: initiation, elongation, and termination

• mono-cistonic mRNA: one mRNA encodes a single protein

  • different from prokaryotes who have poly-cistonic mRNAs

What step is pivotal for viruses ?

initiation

Open Reading Frames (ORFs)

• created when introns are removed from pre-mRNA

• series of triplets coding for amino acids

• end at termination codons

  • UAA, UGA, UAG

Cap-dependent initiation of translation

• most eukaryotic mRNAS use this pathway

• viruses use the cells usual process for reading RNA starting at the cap on RNA

• 48S initiation complex

• 40S ribosome subunit + eukaryotic translation initiation factors (eiF)

Cap-independent initiation of translation

• translation occurs without the need for 5’ cap structure

• ribosome is recruited directly to mRNA via (IRES) Internal ribosome entry site

• ribosomes bind near start codon

• often used under stressful conditions

IRES

Internal ribosome entry site

• some viruses like polio use an IRRS to bypass cap-dependent translation host requirment

Leaky scanning

• occurs when the 40s subunit bypasses the first AUG to initiate translation further downstream

• allows for multiple viral proteins to be synthesized from a single mRNA

• essentially forces 40s subunit to miss a start codon and create multiple viral proteins

Ribosomal Frameshifting

• HIV Gag and Pol ORFs overlap

• most elongating ribosomes terminate translation at the end of Gag ORF producing Gag protein

• Some elongating ribosomes pause and the mRNA slips backwards by 1 nucleotide to be translated in the -1 reading frame producing the longer Gag-Pol protein

• allows translation of downstream ORFs

Translation readthrough by suppressing termination codons

• cell ignores “stop signs” in the RNA and makes longer proteins

• most elongating ribosomes terminate at UAG

• some ribosomes continue translation in the same reading frame

• produces longer readthrough protein

  • encodes more viral genes

  • ends in production of a virion

• ex. TMV

Subgenomci mRNAs

• are shorter RNA molecules derived from full-length genomic RNA of a virus

• ONLY get synthesized by viral RdRP

  • RdRP is an enzyme made by the virus that makes new RNA strands

• 3’ coterminal with the viral genomic RNA meaning same 3’ end as original full length RNA

• act as mRNA to express downstream ORFs

• TMV and SARS-CoV-2

Post-Translational processing of eukaryotic proteins

• proteolytic cleavage to produce mature proteins

  • makes large protein into smaller protein

  • allows viruses to save space by encoding multiple proteins in a single mRNA

  * allows multiple viral proteins to be synthesized from a single mRNA (ex. poliovirus/zika virus/ SARS)

Why do viruses use the host’s protein synthesis machinery ?

viral genomes do not have the capacity to contain all genes necessary to synthesize proteins

Plasma Membrane

• phospholipid bilayer

• intergral and peripheral proteins

enveloped viruses

• steal part of host cell membrane for a protective envelope

Entry of animal viruses is via

endocytic pathways

Plant viruses entry

through wounding

Endocytic pathways

1. Phagocytosis (“cell eating”)

2. Pinocytosis (“cell drinking”)

3. Macropinocytosis (growth factor induced, actin-dependent pinocytosis)

4. Receptor-mediated endocytosis

Key Steps of the animal viral replication cycle (3)

1. Attachment

   • cell surface receptors & coreceptors (host range) to recognize viral protein of virions

2. Entry

   • via endocytic pathways

3. Uncoating (disassembly)

   • removal or degradation of the capsid to release genome into host cell

   • virus breaks open and releases genetic material

4. Genome Replication and Gene Expression

5. Assembly

   • all components of virus assembled into stable particles

6. Maturation

   • stage in life cycle of virus when it becomes infectious

   • viral or cellular proteases often involved

7. Egress

   • newly formed virions release upon lysis

   • some viruses produce enzyme during replication that destroys cellular receptos so newly assembled virions don’t get stuck when exiting cell

DNA Viruses

• DNA replication occurs in host nucleus

• use host viral enzymes for DNA replication and RNA transcription

• stimulate infected cells to enter S phase

  • part of the cell cycle where DNA is naturally replicated

  • virus can continue to produce virions as long as possible if stuck in S phase

RNA viruses

• replicate in the cytoplasm

• all encode their own RNA-dependent RNA Polymerase (RdRP) for RNA synthesis

  • since human cells don’t naturally copy RNA from RNA

• genomic RNA is infectious ONLY for (+) stranded viruses

  • read directly since their genomic RNA acts as mRNA and is directly read by ribosomes to make proteins

  • - and ds RNA must copy their genomes into complementary + strands using RdRP first

• virions contain RdRP except for (+) strand RNA viruses

• error-prone RNA replication because RdRPs lack proof-reading ability except for large coronaviruses

  • use leaky translation

dsDNA Viruses

• use cells nucleus for DNA replication and RNA transcription

ssDNA Viruses

• must turn linear ssDNA to dsDNA form

  • we dont allow ss

• host cells do not contain any DNA-dependent RNA polymerase to convert ssDNA into mRNA

dsRNA viruses

• carry own RNA-dependent RNA polymerase (RdRP) within virion particle

+ssRNA viruses

• translated directly using host cell protein syntheiss machinery

  • RNA in the virus particle functions as mRNA

• carry an RNA-dependent RNA polymerase (RdRP) for replication

  • not needed for translation only replication

-ssRNA viruses

• encode and carry their own RNA-dependent RNA polymerase (RdRP)

  • needed for both transcription and replication

• transcribes -ssRNA genome into +ssRNAs

• synthesizes viral progeny genome

+ssRNA genomes using dsDNA intermediate

• Retroviruses

• unique biology

  • reverse transcription

• integration of viral complementary DNA (cDNA)

viral factories (viroplasm)

may serve as way for viruses to hide from host cell antiviral defense